User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/01

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06/01/11

  • ✓ Cultures/minipreps/digests: assemblies from 5/31/11 (2 each)
  • ✓ Assemblies: re-do KAH188, 190; new KAH191, 195, 196, 197, 198



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH185 = 542
3,4. KAH186 = 2025
5,6. KAH187 = 2269
7,8. KAH188 = 2124
9,10. KAH189 = 2296
11,12. KAH190 = 2368
13,14. KAH192 = 2461
15,16. KAH193 = 2788
17,18. KAH194 = 3032
File:KAH 060111 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.


--> Re-do KAH188, 190 (to get better cloning ratio, less background, use less vector)


Assemblies

  1. KAH188: KAH83/(E/S)/443 ✓ + KAH182/(E/X)/~4875 ✓
  2. KAH190: KAH85/(E/S)/687 ✓ + "
  3. KAH191: (1) KAH185/(E/S)/size + "
  4. KAH195: (2) KAH193/(S/P)/~5988 + polyA2/(X/P)/225 ✓
  5. KAH196: " + KAH171/(X/P)/418 ✓
  6. KAH197: (3) KAH194/(S/P)/~6232 + polyA2/(X/P)/225 ✓
  7. KAH198: " + KAH171/(X/P)/418 ✓

> Digests (Fermentas FD)
--> Specific notes

Reagent Volume   File:KAH 060111 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH185 (E/S) 0.198 2.03 9.9
2. KAH193 (S/P) 0.188 1.74 9.4
3. KAH194 (S/P) 0.178 1.99 8.9


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH83(E/S)/443, 1 ng + KAH182(E/X)/4875, 5 ng KAH188 2:1 (Pick 2)
2. KAH85(E/S)/687, 2 ng + " KAH190 2:1 (Pick 2)
3. KAH85(E/S)/687, 2 ng + " KAH191 1:1 (Pick 4)
4. KAH182(E/X)/ 5 ng lots of colonies, even with only 0.5 DNA
5. polyA2(X/P)/225, 1 ng + KAH193(S/P)/5988, 10 ng KAH195 >10:1 (Pick 2)
6. KAH171/(X/P)/418, 1 ng + " KAH196 >10:1 (Pick 2)
7. KAH193(S/P)/ 5 ng  
5. polyA2(X/P)/225, 1 ng + KAH194(S/P)/6232, 10 ng KAH197 >10:1 (Pick 2)
6. KAH171/(X/P)/418, 1 ng + " KAH198 >10:1 (Pick 2)
7. KAH193(S/P)/ 5 ng  
  1 2 3 4 5 6 7 8 9 10
Insert DNA 3.5 3.5 3.5 --- 2.0 2.0 --- 2.0 2.0 ---
Vector DNA 0.5 0.5 0.5 0.5 2.0 2.0 2.0 2.0 2.0 2.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- --- 3.5 --- --- 2.0 --- --- 2.0
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Add total lign. to 30 μL DH5α-turbo